Azidodifluoromethanide (N3CF2-): In Situ Generation and Nucleophilic Addition to Aldehydes.

A facile one-pot approach for the azidodifluoromethylation of aldehydes via in situ-generated azidodifluoromethenide (N3CF2-) utilizing commercially available TMSCF2Br and NaN3 is disclosed. The formed O-silyl ether products are obtained in yields of up to 91% in short reaction times at ambient temperature. Examples of both inter- and intramolecular [3 + 2] azide-alkyne cycloaddition reactions of the installed azidodifluoromethyl handles are also presented, demonstrating the prospective synthetic and biochemical functionality and utility.

Addition of Imidazolium-Based Ionic Liquid to Improve Methanol Production in Polyamine-Assisted CO2 Capture and Conversion Systems Using Pincer Catalysts.

Ionic liquids have been studied as CO2 capture agents. However, they are rarely used in combined CO2 capture and conversion processes. Utilizing imidazolium-based ionic liquids, the conversion of CO2 to methanol was greatly improved in polyamine assisted systems catalyzed by homogeneous pincer catalysts with Ru and Mn metal centers. Among the ionic liquids tested, [BMIM]OAc was found to perform the best under the given reaction conditions. Among the polyamine tested, pentaethylenehexamine (PEHA) led to the highest conversion rates. Ru-Macho and Ru-Macho-BH were the most active catalysts. Direct air capture utilizing PEHA as the capture material was also demonstrated and produced an 86 % conversion of the captured CO2 to methanol in the presence of [BMIM]OAc.

Integrated Carbon Dioxide Capture and Conversion to Methanol Utilizing Tertiary Amines over a Heterogenous Cu/ZnO/Al2O3 Catalyst.

Increasing carbon dioxide emissions has sparked a growing interest in capturing these emissions at the source of their release. For such processes, amines can be used as carbon dioxide capture agents. Herein, CO2 was captured under ambient conditions using solutions of amines and polyamines in ethylene glycol. The captured solutions were then successfully hydrogenated to methanol under hydrogen pressure with a heterogeneous Cu/ZnO/Al2O3 industrial catalyst. An extensive amine scope found that tetramethyl-1,6-hexanediamine, with two tertiary amine sites, provided the highest methanol productivity. This reaction was then optimized to achieve up to 89% methanol yield under relatively mild conditions of 250 °C and 80 bar H2 pressure. The catalyst was shown to be recyclable over five reaction cycles.

Appearance of aseptic vascular grafts after endovascular aortic repair on [(18)F]fluorodeoxyglucose positron emission tomography/computed tomography.

BACKGROUND: Diagnosis of prosthetic vascular graft infection with [(18)F]fluorodeoxyglucose positron emission tomography/computed tomography (18F-FDG PET/CT) allows for early detection of functional changes associated with infection, based on increased glucose utilization by activated macrophages and granulocytes. Aseptic vascular grafts, like all foreign bodies, can stimulate an inflammatory response, which can present as increased activity on 18F-FDG PET/CT. Consequently, distinguishing aseptic inflammation from graft infection, though important, can be difficult. In the case of endovascular aneurysm repair (EVAR), a minimally invasive procedure involving the transfemoral insertion of an endoprosthetic stent graft, the normal postoperative appearance of these grafts on 18F-FDG PET/CT can vary over time, potentially confounding study interpretation. AIM: To investigate the visual, semiquantitative, and temporal characteristics of aseptic vascular grafts in patients status post EVAR. METHODS: In this observational retrospective cohort study, patients with history of EVAR who underwent 18F-FDG PET/CT for indications other than infection were identified retrospectively. All patients were asymptomatic for graft infection - no abdominal pain, fever of unknown origin, sepsis, or leukocytosis - at the time of imaging and for ≥ 2 mo after each PET/CT. Imaging studies such as CT for each patient were also reviewed, and any patients with suspected or confirmed vascular graft infection were excluded. One hundred two scans performed on 43 patients (34 males; 9 females; age = 77 ± 8 years at the time of the final PET/CT) were retrospectively reviewed. All 43 patients had an abdominal aortic (AA) vascular graft, 40 patients had a right iliac (RI) limb graft, and 41 patients had a left iliac (LI) limb graft. Twenty-two patients had 1 PET/CT and 21 patients had from 2 to 9 PET/CTs. Grafts were imaged between 2 mo to 168 mo (about 14 years) post placement. Eight grafts were imaged within 6 mo of placement, including three that were imaged within three months of placement. The mean interval between graft placement and PET/CT for all 102 scans was 51 ± 39 mo. PET/CT data was reconstructed with region-of-interest analysis of proximal, mid and distal portions of the grafts and background ascending aorta. Maximum standardized uptake value (SUVmax) was recorded for each region. SUVmax-to-background uptake ratios (URs) were calculated. Visual assessment was performed using a 2-pattern grading scale: Diffuse (homogeneous uptake less than liver uptake) and focal (one or more areas of focal uptake in any part of the graft). Statistical analysis was performed. RESULTS: In total, there were 306 AA grafts, 285 LI grafts, 282 RI grafts, and 306 ascending aorta background SUVmax measurements. For all 102 scans, mean SUVmax values for AA grafts were 2.8-3.0 along proximal, mid, and distal segments. Mean SUVmax values for LI grafts and RI grafts were 2.7-2.8. Mean SUVmax values for background were 2.5 ± 0.5. Mean URs were 1.1-1.2. Visual analysis of the scans reflected results of quantitative analysis. On visual inspection, 98% revealed diffuse, homogeneous 18F-FDG uptake less than liver. Graft URs and visual pattern categories were significantly associated for AA graft URs (F-ratio = 21.5, P < 0.001), LI graft URs (F-ratio = 20.4, P < 0.001), and RI graft URs (F-ratio = 30.4, P < 0.001). Thus, visual patterns of 18F-FDG uptake corresponded statistically significantly to semiquantitative URs. The age of grafts showing focal patterns was greater than grafts showing diffuse patterns, 87 ± 89 vs 50 ± 37 mo, respectively (P = 0.02). URs were significantly associated with graft age for AA grafts (r = 0.19, P = 0.001). URs were also significantly associated with graft age for LI grafts (r = 0.25, P < 0.0001), and RI grafts (r = 0.31, P < 0.001). Quartiles of similar numbers of graft (n = 25-27) grouped by graft age indicated that URs were significantly higher for 4th quartile vs 2nd quartile URs (F-ratio = 19.5, P < 0.001). When evaluating URs, graft SUVmax values within 10%-20% of the ascending aorta SUVmax is evident in aseptic grafts, except for grafts in the oldest quartiles. In this study, grafts in the oldest quartiles (> 7 years post EVAR) showed SUVmax up to 30% higher than the ascending aorta SUVmax. CONCLUSION: Characteristics of an aseptic vascular stent graft in the aorta and iliac vessels on 18F-FDG PET/CT include graft SUVmax values within 10%-20% of the ascending aorta background SUVmax. The SUVmax of older aseptic grafts can be as much as 30% above background. The visual uptake pattern of diffuse, homogeneous uptake less than liver was seen in 98% of aseptic vascular grafts, making this pattern particularly reassuring for clinicians.

Bridging the gap: The critical role of laboratory developed tests in clinical toxicology.

•Toxicology testing provides valuable information for patient management.•Current in vitro diagnostics (IVDs) are unable to meet all clinical needs.•Lab-developed tests (LDTs) in toxicology can be used to close clinical care gaps.•LDTs in clinical toxicology are almost exclusively mass spectrometry-based methods.

Resolving icteric interference by a novel photoisomerization device: Bilibox.

Icterus, a phenomenon caused by bilirubin elevation in the blood, is a common endogenous interference in chemistry testing, occurring either spectrally or through chemical reactivity with assay reagents. Often, laboratories have few options other than to dilute or reject samples exceeding icteric thresholds. However, recent studies have optimized in vitro photoisomerization of bilirubin to a 17-minute bilirubin half-life using 500 nm light at 37 °C. Using an enzymatic creatinine assay as a model, due to its prevalence in routine laboratory testing and susceptibility to icteric interference, our study explores the usage of in vitro photoisomerization by replicating these conditions in a device, the Bilibox, as means of resolving icterus in laboratory testing. Left-over icteric and non-icteric clinical samples, collected by lithium heparin vacutainer (n = 10), were analyzed for baseline creatinine, diluted creatinine (1:4 0.9 % NaCl), total bilirubin, direct bilirubin, and hemolysis, icterus and lipemia (HIL) indices. Samples were then placed in the Bilibox in two intervals of 30 min with repeat measurements of the aforementioned analytes. On average, icteric-index, total bilirubin (TBIL), and direct bilirubin (DBIL) decreased by 33.5, 39.1 and 39.9 % respectively following 30 min of Bilibox treatment; and by 47.6 %, 63.7 % and 59.8 % following 60 min. The average percent difference between the pre-exposure diluted and undiluted creatinine was 5.8 %, demonstrating the icterus interference. Following Bilibox treatment, the difference between undiluted (post-exposure) and diluted (pre-exposure) creatinine decreased to 0.02 % (p = 0.0232) and 2.2 % (p = 0.0021) at 30 and 60 min of treatment respectively, demonstrating resolution of interference. Consequently, photoisomerization can be utilized as an additional and reasonably quick method for resolving icterus when dilutional methods cannot be applied.

gem-Halofluorocyclopropanes via [2 + 1] Cycloadditions of In Situ Generated CFX Carbene with Alkenes.

An efficient and operationally simple synthesis of gem-bromofluorocyclopropanes under mild conditions has been developed. The method employs ethyl dibromofluoroacetate (EDBFA) as an accessible and inexpensive source of the bromofluorocarbene (:CFBr) intermediate. The protocol provides the bromofluorocyclopropane products in excellent yields, including examples synthesized in multigram scales. The chlorinated ester, ethyl dichlorofluoroacetate (EDCFA), is also utilized to make the analogous gem-chlorofluorocyclopropanes.

Development of a clinical polygenic risk score assay and reporting workflow.

Implementation of polygenic risk scores (PRS) may improve disease prevention and management but poses several challenges: the construction of clinically valid assays, interpretation for individual patients, and the development of clinical workflows and resources to support their use in patient care. For the ongoing Veterans Affairs Genomic Medicine at Veterans Affairs (GenoVA) Study we developed a clinical genotype array-based assay for six published PRS. We used data from 36,423 Mass General Brigham Biobank participants and adjustment for population structure to replicate known PRS-disease associations and published PRS thresholds for a disease odds ratio (OR) of 2 (ranging from 1.75 (95% CI: 1.57-1.95) for type 2 diabetes to 2.38 (95% CI: 2.07-2.73) for breast cancer). After confirming the high performance and robustness of the pipeline for use as a clinical assay for individual patients, we analyzed the first 227 prospective samples from the GenoVA Study and found that the frequency of PRS corresponding to published OR > 2 ranged from 13/227 (5.7%) for colorectal cancer to 23/150 (15.3%) for prostate cancer. In addition to the PRS laboratory report, we developed physician- and patient-oriented informational materials to support decision-making about PRS results. Our work illustrates the generalizable development of a clinical PRS assay for multiple conditions and the technical, reporting and clinical workflow challenges for implementing PRS information in the clinic.

Rapid serum clot tubes reduce haemolysis due to pneumatic tube transport.

AIMS: Pneumatic tube systems (PTSs) are critical for modern hospital operations, allowing for rapid sample transport. Despite widespread use, PTSs can compromise specimen integrity and affect laboratory values. Our objective was to prove that rapid serum clot tubes (RST) provide protective benefits over plasma during PTS transport and can be a practical solution for certain PTS routes. METHODS: In this study, we compared the effects of PTS transport on cell lysis indicators: h-index, lactate dehydrogenase (LDH) and potassium (K+), in RST versus lithium heparin gel separator tubes using 10 volunteers. RESULTS: In comparison with plasma, RST showed a median reduction in PTS-induced haemolysis of 80.4% (p=0.0049), with a reduction in post-PTS median LDH concentration (49.7%, p=0.04) and K+ concentration (50.0%, p=0.0273). CONCLUSION: This study demonstrates RST tubes can significantly reduce PTS-induced haemolysis and can be recommended for poor PTS routes.

Automated Pharmacogenomic Reports for Clinical Genome Sequencing.

Clinical laboratories offering genome sequencing have the opportunity to return pharmacogenomic findings to patients, providing the added benefit of preemptive testing that could help inform medication selection or dosing throughout the lifespan. Implementation of pharmacogenomic reporting must address several challenges, including inherent limitations in short-read genome sequencing methods, gene and variant selection, standardization of genotype and phenotype nomenclature, and choice of guidelines and drugs to report. An automated pipeline, lmPGX, was developed as an end-to-end solution that produces two versions of a pharmacogenomic report, presenting either Clinical Pharmacogenetics Implementation Consortium or US Food and Drug Administration guidelines for 12 genes. The pipeline was validated for performance using reference samples and pharmacogenetic data from the Genetic Testing Reference Materials Coordination Program. To determine performance and limitations, lmPGX was compared with three additional publicly available pharmacogenomic pipelines. The lmPGX pipeline offers clinical laboratories an opportunity for seamless integration of pharmacogenomic results with genome reporting.

The ALT upper reference interval debate: Blame it on the alcohol.

BACKGROUND: In 2017, the American College of Gastroenterology (ACG) and the North American Society of Pediatric Gastroenterology, Hepatology and Nutrition published clinical guidelines recommending the use of alanine aminotransferase (ALT) upper reference limits (URL) of 33, 25, 26, and 22 U/l for men, women, boys, and girls, respectively. This was opposed by laboratory experts who advocated for the use of higher URL of 59, 41, 33, and 24 U/l instead. We suspected that the variable inclusion of individuals who consumed alcohol to be a major contributing source of URL variability and debate. METHODS: Outpatient ALT data (n = 7379) were collected from unique individuals ≥13 y with BMIs of ≥19 and ≤25. A total of 222 (3%) were excluded due to suspected liver disease. Patients were split into a pediatric group (age 13-17 y), an alcohol-restricted adult group (age 18-20 y), and adults with access to alcohol by decade (i.e., age 21-29, 30-39, 40-49, 50-59, 60-69, 70-79, and ≥ 80 y). All ALT values were measured on Roche Cobas 8000 with pyridoxal phosphate and traceable to the IFCC-reference measurement procedure. RESULTS: We derived URL similar to CALIPER for our pediatric population, but closer to ACG-proposed URL in our alcohol-restricted adult group. The URL increased significantly in men and women for all other age groups. CONCLUSIONS: The discrepancy in ALT URL in clinical laboratories may be attributable in part due to the variable inclusion of individuals who recently consumed alcohol in local population derivation studies.

Reanalysis of eMERGE phase III sequence variants in 10,500 participants and infrastructure to support the automated return of knowledge updates.

PURPOSE: The clinical genomics knowledgebase is dynamic with variant classifications changing as newly identified cases, additional population data, and other evidence become available. This is a challenge for the clinical laboratory because of limited resource availability for variant reassessment. METHODS: Throughout the Electronic Medical Records and Genomics phase III program, clinical sites associated with the Mass General Brigham/Broad sequencing center received automated, real-time notifications when reported variants were reclassified. In this study, we summarized the nature of these reclassifications and described the proactive reassessment framework we used for the Electronic Medical Records and Genomics program data set to identify variants most likely to undergo reclassification. RESULTS: Reanalysis of 1855 variants led to the reclassification of 2% (n = 45) of variants, affecting 0.6% (n = 67) of participants. Of these reclassifications, 78% (n = 35) were high-impact changes affecting reportability, with 8 variants downgraded from likely pathogenic/pathogenic to variants of uncertain significance (VUS) and 27 variants upgraded from VUS to likely pathogenic/pathogenic. Most upgraded variants (67%) were initially classified as VUS-Favor Pathogenic, highlighting the benefit of VUS subcategorization. The most common reason for reclassification was new published case data and/or functional evidence. CONCLUSION: Our results highlight the importance of periodic sequence variant reevaluation and the need for automated approaches to advance routine implementation of variant reevaluations in clinical practice.

Returning actionable genomic results in a research biobank: Analytic validity, clinical implementation, and resource utilization.

Over 100 million research participants around the world have had research array-based genotyping (GT) or genome sequencing (GS), but only a small fraction of these have been offered return of actionable genomic findings (gRoR). Between 2017 and 2021, we analyzed genomic results from 36,417 participants in the Mass General Brigham Biobank and offered to confirm and return pathogenic and likely pathogenic variants (PLPVs) in 59 genes. Variant verification prior to participant recontact revealed that GT falsely identified PLPVs in 44.9% of samples, and GT failed to identify 72.0% of PLPVs detected in a subset of samples that were also sequenced. GT and GS detected verified PLPVs in 1% and 2.5% of the cohort, respectively. Of 256 participants who were alerted that they carried actionable PLPVs, 37.5% actively or passively declined further disclosure. 76.3% of those carrying PLPVs were unaware that they were carrying the variant, and over half of those met published professional criteria for genetic testing but had never been tested. This gRoR protocol cost approximately $129,000 USD per year in laboratory testing and research staff support, representing $14 per participant whose DNA was analyzed or $3,224 per participant in whom a PLPV was confirmed and disclosed. These data provide logistical details around gRoR that could help other investigators planning to return genomic results.

Determination of 97.5th and 99th percentile upper reference limits for heart-type fatty acid-binding protein (H-FABP) in a US population.

BACKGROUND: Heart-type fatty acid binding protein (H-FABP), a low molecular weight protein found primarily in myocardial tissue, has been identified as a potential biomarker in the detection of acute coronary syndrome and acute kidney injury. To further investigate clinical utility, we sought to establish an upper reference limit (URL) of H-FABP within a healthy U.S. METHODS: Serum samples of healthy donors were acquired from the AACC Universal Sample Bank. We analyzed 355 samples for H-FABP concentration using the Randox Laboratories immunoturbidimetric assay on the Roche Cobas 8000 series analyzer. RESULTS: The final sample population consisted of individuals aged 18-74 y, with 170 males and 185 females. The distribution of the population exhibited a strong positive skew, affecting outlier analysis and URL determination. The 97.5th-percentile URL was found to be 7.4 ng/ml (95% CI: 6.3-9.2), while the 99th-percentile URL was 12.1 ng/ml (8.6-14.9). CONCLUSION: As the URL for H-FABP is highly affected by population distribution and outlier removal, final determination for an assay cutoff should be made in the context of clinical utility, either as a standalone assay or in conjunction with other biomarkers, and the desired clinical sensitivity and specificity.

Identification of novel ADAMTS1, ADAMTS4 and ADAMTS5 cleavage sites in versican using a label-free quantitative proteomics approach.

The chondroitin sulfate proteoglycan versican is important for embryonic development and several human disorders. The versican V1 splice isoform is widely expressed and cleaved by ADAMTS proteases at a well-characterized site, Glu441-Ala442. Since ADAMTS proteases cleave the homologous proteoglycan aggrecan at multiple sites, we hypothesized that additional cleavage sites existed within versican. We report a quantitative label-free approach that ranks abundance of liquid chromatography-tandem mass spectrometry (LC-MS/MS)-identified semi-tryptic peptides after versican digestion by ADAMTS1, ADAMTS4 and ADAMTS5 to identify site-specific cleavages. Recombinant purified versican V1 constructs were digested with the recombinant full-length proteases, using catalytically inactive mutant proteases in control digests. Semi-tryptic peptide abundance ratios determined by LC-MS/MS in ADAMTS:control digests were compared to the mean of all identified peptides to obtain a z-score by which outlier peptides were ranked, using semi-tryptic peptides identifying Glu441 -Ala442 cleavage as the benchmark. Tryptic peptides with higher abundance in control digests supported cleavage site identification. We identified several novel cleavage sites supporting the ADAMTS1/4/5 cleavage site preference for a P1-Glu residue in proteoglycan substrates. Digestion of proteins in vitro and application of this z-score approach is potentially widely applicable for mapping protease cleavage sites using label-free proteomics. SIGNIFICANCE: Versican abundance and turnover are relevant to the pathogenesis of several human disorders. Versican is cleaved by A Disintegrin-like And Metalloprotease with Thrombospondin type 1 motifs (ADAMTS) family members at Glu441-Ala442, generating a bioactive proteoform called versikine, but additional cleavage sites and the site-specificity of individual ADAMTS proteases is unexplored. Here, we used a label-free proteomics strategy to identify versican cleavage sites for 3 ADAMTS proteases, applying a novel z-score-based statistical approach to compare the protease digests of versican to controls (digests with inactive protease) using the known protease cleavage site as a benchmark. We identified 21 novel cleavage sites that had a comparable z-score to the benchmark. Given the functional significance of versikine, they represent potentially significant cleavages and helped to refine a substrate site preference for each protease.The z-score approach is potentially widely applicable for discovery of site-specific cleavages within an purified protein or small ensemble of proteins using any protease.